A typical lager sweet wort consists of a complex mixture of starch derived carbohydrates, which are classified as fermentable or non-fermentable according to whether they can be converted into ethanol by brewer's yeast. The fermentable carbohydrates are formed by hydrolysis of grain starches by two enzymes, .alpha. and .beta.-amylase, derived from malted barley. In most American lagers the malted barley also serves as the predominant starch source while a smaller proportion is derived from nondiastatic adjunct grains. In the U.S., corn grits, #4 brewer's rice and various corn syrups are the predominant adjuncts.
All grain starches are glucose homopolymers in which the glucose residues are linked by either .alpha.-1,4 or .alpha.-1,6 bonds. During the mashing cycle the starches are first solubilized and then a portion of the solubilized large starch molecules are hydrolyzed to non-fermentable dextrins and to three low molecular weight sugars which brewer's yeast can ferment to ethyl alcohol. The major fermentable sugars are glucose, maltose, and maltotriose while traces of sucrose and fructose are also present.
The non-fermentable or limit dextrin fraction consists of all sugars of a higher degree of polymerization (DP) than maltotriose. The bulk of the limit dextrins appear to be quite large since most are retained by diafiltration membranes which are able to pass molecules .ltoreq.10,000 daltons.
As indicated above, the hydrolysis of the grain starches is catalyzed by two amylases endogenous to malted barley. One, .alpha.-amylase, is an endoamylase which randomly cleaves .alpha.-1,4 bonds in the interior of the raw, largely insoluble starch molecules, fragmenting them into large but soluble polysaccharides termed dextrins. The second, .beta.-amylase, is an exo-amylase which sequentially cleaves .alpha.-1,4 bonds from the non-reducing end of these dextrins producing the three fermentable sugars described above. Both enzymes are inactive towards the .alpha.-1,6 linkages (branch points) of the starches (i.e. they are unable to debranch the starch molecule) and this results in the formation of the forementioned limit dextrins.
The composition of the wort can vary depending on starting materials, mash cycle and other variables. The carbohydrate composition of a typical wort consists of 65-80% fermentable sugars, and limit dextrins ranging from 20-35%. At the end of fermentation, the fermentable fraction is converted to ethanol at a final concentration ranging from about 3 to about 6% w/w. The limit dextrins are not converted during fermentation and form the majority of the dissolved solids (termed real extract) in the final beer.
Recently, reduced calorie beers have become popular in the U.S. beer market. These beers may be formulated by: (1) reducing both the alcohol and real extract concentrations in the beer to attain the desired calorie level, or (2) by hydrolyzing the limit dextrins to fermentable sugars with exogenous enzymes, one component of which is capable of debranching the limit dextrins. The latter method is advantageous since it allows one to attain the desired calorie level with minimum reduction of the alcohol content of the packaged product. The exogenous enzyme most commonly used to hydrolyze the limit dextrins is glucoamylase, a nonspecific exoamylase derived from a variety of fungal sources e.g. Aspergillus niger, Rhizopus delemar, etc. (Pazur, J. Methods of Enzymology, XVIII (ed. Ginsberg, V.), Academic Press (1975), p. 931).
Glucoamylase is active vs. both .alpha.-1,4 and .alpha.-1,6 linkages and therefore is capable of hydrolyzing starch to glucose. It attacks the starch molecule from the nonreducing end producing glucose as the sole end product. It is also active vs. starch derived oligosaccharides, e.g. maltose, maltotriose, isomaltose, etc.
In theory debranching enzymes, such as glucoamylase, may be added at any time during the brewing process. In practice brewers prefer to add them in fermentation because the fermentation process itself requires 6-15 days depending on pitching rate, fermentation, temperature, etc. In contrast, the brewhouse operations are of much shorter duration (2-4 hrs/brew) and it operates under tight scheduling constraints. Therefore, these enzymes are employed as fermentation adjuncts as taught by Gablinger in U.S. Pat. No. 3,379,534, and the limit dextrins are hydrolyzed to fermentable sugars, which the yeast convert to ethanol. Operationally these beers ferment to a lower specific gravity because of (1) increased alcohol, and (2) decreased real extract as compared to the same beer prepared without the use of exogenous enzymes. Such beers are referred to as superattenuated beers.
In contrast to European brewers, U.S. brewers have always derived substantial quantities of wort extract (fermentable and non-fermentable carbohydrates) from sources other than malted barley. These sources are termed adjuncts and consist for the most part of ungerminated cereal grains termed cereal adjuncts, and corn syrups supplied commercially by the corn wet milling industry, termed liquid adjuncts. The cereal adjuncts most commonly employed by U.S. brewers are corn grits and broken polished rice. U.S. brewers typically derive from 30-50% of the wort extract from these adjuncts.
Cereal adjuncts are processed simultaneously with malt using a double mash upward infusion system. Typically the cereal adjunct is added to a vessel called the cereal cooker which contains a charge of malt ranging from 10-20% w/w of that of the adjunct. The cooker mash is rested at temperatures ranging from 100.degree.-120.degree. F. for 20-40 minutes. The mash is then raised to a vigorous boil in about 50-60 minutes and held there for 20-45 minutes. During the rise the mash gels as the starch imbibes water and then liquifies as the malt enzymes, particularly .alpha.-amylase, causes the swollen starch granules to rupture and disperse throughout the mash.
As the contents of the cooker mash are being boiled the majority of the malt is charged into a second larger vessel called the mash tun. The malt mash is rested at 100.degree.-120.degree. F. to extract the diastases for a period ranging from 15-30 minutes, the end of which coincides precisely with the end of boiling of the cereal cooker mash. The contents of the cereal cooker are then transferred to the mash tun so that the combined contents of the mash tun are elevated to the conversion temperature. The combined mash is held at the desired conversion temperature (150.degree.-160.degree. F.) for a fixed period of time (40-90 minutes). During the rise to and rest at conversion temperature the malt enzymes hydrolyze the liquified adjunct and malt starches to the three fermentable sugars described above. At the end of the conversion period, the contents are mashed off, i.e. heat is applied to raise the mash tun contents to temperatures ranging from 170.degree.-185.degree. F., which serves to inactivate the malt enzymes and to facilitate the subsequent filtration operation (lautering or mash filter) which yields the clarified sweet wort.
In contrast to cereal adjuncts, liquid adjuncts are commonly added to the clarified wort at the kettle. The most commonly used liquid adjuncts are starch-derived syrups, such as corn syrups, which have low to moderate dextrose equivalents (D.E.) of about 30 to about 65. The use of liquid adjuncts offers several advantages (Bradee, L., in "The Practical Brewer" H. Broderick (ed.) published by the Master Brew. Assoc. of Americas, 1978):
(1) All-malt mashes with liquid adjuncts run off faster than double infusion mashes with cereal adjuncts. This permits the production of more brews in the same period of time which in turn increases the capacity of a normal brewhouse with limited capital investment.
(2) Liquid adjuncts facilitate high gravity brewing. This increases the production capacity of the brewery with no capital investment.
(3) Quantitative extract yields are obtained from liquid adjuncts. In comparison substantial losses are incurred during the lautering operation of double infusion mashes.
(4) Liquid adjuncts are easier to sanitize, store and handle. Their use permits the elimination of cereal adjunct grain equipment (storage bins, conveyors, mills, scales) which are expensive and sources of infestation.
(5) A wide variety of liquid adjuncts are available from commercial corn wet millers. The brewer is thus able to use these products to vary the composition of the wort over a wide range.
(6) The compositions of liquid adjuncts can be controlled within very narrow specifications. This assures the brewer of uniformity of the adjunct ingredient.
Liquid adjuncts are produced by three basic processes:
(1) The acid process in which starch is liquified and saccharified with acid at elevated temperatures.
(2) The acid-enzyme process in which starch is liquified with acid at elevated temperatures followed by saccharification with glucogenic or maltogenic enzymes or mixtures thereof under pH and temperature conditions favorable to the enzymes.
(3) The double enzyme process in which starch is liquified at high temperature with a thermostable endoamylase and then saccharified with glucogenic or maltogenic enzymes as above.
The latter two methods have largely displaced the acid process since: (1) they are less destructive to the substrate, (2) they are much more specific, and (3) allow for the precise formulation of a wide variety of syrups depending on the enzyme systems employed. Currently most brewer's liquid adjuncts are produced by the acid-enzyme procedure.
Fungal glucoamylase does not produce completely superattenuated beer from wort containing low or moderate D.E. (dextrose equivalent) starch-derived syrups. The low or moderate D.E. starch-derived syrups apparently contain a type of dextrin which is not present in cereal adjuncts and which is not hydrolyzed by fungal glucoamylase.
The carbohydrases produced by Schwanniomyces sp. have been studied by many investigators. There are two extracellular amylases secreted by yeast of Schwanniomyces sp. These are an .alpha.-amylase which has the ability to hydrolyze the .alpha.1,4 linkages of starch and a glucoamylase type debranching enzyme which has the ability to hydrolyze not only the .alpha.-1,4 linkages of starch, but also has the ability to hydrolyze the .alpha.-1,6 linkages (Oteng-Gyang, K. Moulin, G., Galzy, P., Zeitschrift fur Allgemeine Mikrobiologie, 1981, 21, 537: Wilson, J. J., and Ingeldew, M. W., Appl. and Envir. Micro., 1982, 44, 301). Since Schwanniomyces is capable of secreting these starch degrading enzymes, various workers have used this organism for the production of single cell protein or ethanol from starchy substrates (Oteng-Gyang K., et al. Eur. J. Applied Microbiol. Biotech. 9, 129, 1980; Callegja G., et al. Biotech. Lett. 4, 543, 1982).
Recently Canadian workers (Sills, A., et al. Proceedings of the European Brewing Convention, 1983) have shown that, like fungal glucoamylase, the carbohydrases produced by this yeast may be used to prepare a superattenuated (i.e. reduced carbohydrate content) beer. The carbohydrases employed by these workers contained an .alpha.-amylase in addition to a glucoamylase type debranching enzyme. These workers noted an inverse relation between the time of addition and the degree of superattenuation i.e., delayed addition yielded beers with higher degrees of attenuation.